biochemistry lecture 6. functions of nucleotides and nucleic acids nucleotide functions: –energy...
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Biochemistry
Lecture 6
Functions ofNucleotides and Nucleic
Acids• Nucleotide Functions:
– Energy for metabolism (ATP)– Enzyme cofactors (NAD+)– Signal transduction (cAMP)
• Nucleic Acid Functions: – Storage of genetic info (DNA)– Transmission of genetic info (mRNA)– Processing of genetic information (ribozymes)– Protein synthesis (tRNA and rRNA)
NucleotideNucleosideNucleobase
Pyrimidine Nucleobases
Purine Nucleobases
UV Absorption of Nucleobases
-D-ribofuranose in RNA
-2’-deoxy-D-ribofuranose in DNA
N-Glycosidic Bond
Polynucleotides
Hydrolysis of RNA
Hydrogen Bonding!
Discovery of DNA Structure
• One of the most important discoveries in biology
• Why is this important– "This structure has novel features which are of
considerable biological interest“--- Watson and Crick, Nature, 1953
• Good illustration of science in action:– Missteps in the path to a discovery– Value of knowledge– Value of collaboration– Cost of sharing your data too early
Covalent Structure of DNA (1868-1935)
• Friedrich Miescher isolates “nuclein” from cell nuclei
• Hydrolysis of nuclein:– phosphate– pentose– and a nucleobase
• Chemical analysis:– phosphodiester linkages– pentose is ribofuranoside
O
OH
H HH
Thymine
H
CH2O P
OH
O
O
P OOH
OH
O
H
H HH
Adenine
H
CH2O P
OH
O
O
Structure of DNA: 1929
(Levene and London)
Structure of DNA:
1935(Levene and Tipson)
C5H7OThymine
O
P
O
POH
OH
O
O
O
OH
C5H7OAdenine
O
Road to the Double Helix• Franklin and Wilkins:
–“Cross” means helix
–“Diamonds” mean
that the phosphate-
sugar backbone
is outside
– Calculated helical
parameters
• Watson and Crick:
– Missing layer means
alternating pattern
(major & minor groove)
– Hydrogen bonding:
A pairs with T
G pairs with C
Double helix fits the data!
Watson, Crick, and Wilkins shared 1962 Nobel Prize
Franklin died in 1958
Other forms of DNA
The Central Dogma
DNA Replication
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”
Watson and Crick, in their Nature paper,1953
Using DNA Structure
Thermal Denaturation
Molecular Mechanisms of Spontaneous Mutagenesis
• Deamination• Very slow reactions• Large number of residues• The net effect is significant: 100 C U events /day in a mammalian cell
• Depurination• N-glycosidic bond is hydrolyzed• Significant for purines: 10,000 purines lost/day in a mammalian cell
• Cells have mechanisms to correct most of these modifications.
DNA Technologies
DNA Cloning
Restriction Enzymes
Antibiotic Selection• Antibiotics, such as penicillin and ampicillin, kill
bacteria
• Plasmids can carry genes that give host bacterium a resistance against antibiotics
• Allows growth (selection) of bacteria that have taken up the plasmid
NH
S
N
N
OO
H
OOH
HH
PCR
PolymeraseChainReaction
Site-Directed Mutagenesis
DNA Electrophoresis
DNA Sequencing
DNA Sequencing
Shotgun Sequencing
DNA Fingerprinting
Expression of Cloned Genes
Protein Purification
Eukaryotic Gene Expression in Bacteria
• An eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium
• Eukaryotic genes have
– Exons: coding regions
– Introns: noncoding regions
• Introns in eukaryouric gene pose problems
• Bacteria cannot splice introns out
• mRNA is intron-free genetic material
cDNA
DNA Microarrays: Applications
DNA Microarrays allow simultaneous screening of many thousands of genes: high-throughput screening
• genome wide genotyping
– Which genes are present in this individual?
• tissue-specific gene expression
– Which genes are used to make proteins?
• mutational analysis
– Which genes have been mutated?
DNA Microarrays: Design Two fundamental approaches• One-color array
– Patented and commerialized by Affymetrix– Photolitographic synthesis of probe DNA on the chip– Targets are biotin labeled– Bound targets detected using streptavidin-fluorofore complex– Widely used in industry
• Two-color array– Developed by Stanford University, 1996– Probes sometimes pipetted on the chip– Targets linked to either green or red fluorescent labels– Used often in academia